Cell culture array system for automated assays and methods of operation and manufacture thereof

ABSTRACT

A number of novel improved microfluidic configurations and systems and methods of manufacture and operation. In one embodiment, three wells are used for independent cell culture systems in a cell culture array. In a second aspect, artificial sinusoids with artificial epithelial barriers are provided with just one (optionally shared or multiplexed) fluidic inlet and one (optionally shared or multiplexed) fluidic output, where the medium output also functions as a cellular input. A pneumatic cell loader combined with other components provides a fully automated cell culture system. Magnetic alignment of plate molds provides advantages and ease of molded manufacture.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from provisional patent application61/037,297 filed Mar. 17, 2008 and from 61/018,882 filed Jan. 3, 2008,each incorporated herein by reference.

This application discusses technology related to U.S. Ser. No.11/994,997, filed Aug. 11, 2008, which is a National Stage Entry ofPCT/US06/26364, filed Jul. 6, 2006 and which claims priority fromprovisional patent application 60/773,467 filed 14 Feb. 2006 and fromprovisional patent application 60/697,449 filed 7 Jul. 2005.

This application discusses technology related to U.S. application Ser.No. 12/019,857, filed Jan. 25, 2008, which claims priority to U.S.Provisional Patent Application No. 60/900,651 filed on Feb. 8, 2007.

This application discusses technology related to U.S. application Ser.No. 11/648,207, filed Dec. 29, 2006, which claims priority to U.S.Provisional Patent Application U.S. provisional patent application No.60/756,399 filed on Jan. 4, 2006. All of these applications areincorporated herein by reference for all purposes.

COPYRIGHT NOTICE

Pursuant to 37 C.F.R. 1.71(e), applicants note that a portion of thisdisclosure contains material that is subject to copyright protection(such as, but not limited to, diagrams, device photographs, or any otheraspects of this submission for which copyright protection is or may beavailable in any jurisdiction). The copyright owner has no objection tothe facsimile reproduction by anyone of the patent document or patentdisclosure, as it appears in the Patent and Trademark Office patent fileor records, but otherwise reserves all copyright rights whatsoever.

FIELD OF THE INVENTION

The invention in various embodiments relates to handling ofmicro-objects, such as cells or micro-fabricated particles are beads,using a microfluidic system and particularly is directed to aconfiguration that can be used with various standard automated handlingsystems. In particular embodiments, the invention involves an automatedsystem for cell culture.

BACKGROUND OF THE INVENTION

The discussion of any work, publications, sales, or activity anywhere inthis submission, including in any documents submitted with thisapplication, shall not be taken as an admission that any such workconstitutes prior art. The discussion of any activity, work, orpublication herein is not an admission that such activity, work, orpublication existed or was known in any particular jurisdiction.

Microfluidic cell culture is a promising technology for applications inthe drug screening industry. Key benefits include improved biologicalfunction, higher-quality cell-based data, reduced reagent consumption,and lower cost. High quality molecular and cellular sample preparationsare important for various clinical, research, and other applications. Invitro samples that closely represent their in vivo characteristics canpotentially benefit a wide range of molecular and cellular applications.Handling, characterization, culturing, and visualization of cells orother biologically or chemically active materials (such as beads coatedwith various biological molecules) has become increasingly valued in thefields of drug discovery, disease diagnoses and analysis, and a varietyof other therapeutic and experimental work.

Mammalian cell culture is particularly challenging, particularly formaintaining effective solid aggregates of cells in culture. Advanceshave been made by adapting various microfabrication and microfluidictechnologies to cell culture, though there remains an ongoing need for adevice that can be economically manufactured and used to provideeffective cell culture.

Publications and/or patent documents that discuss various strategiesrelated to cell culture using microfluidic systems and relatedactivities include the following U.S. patent applications and non-patentliterature, which, along with all citations therein, are incorporatedherein by reference for all purposes. A listing of these references heredoes not indicate the references constitute prior art.

-   Cytoplex, Inc. 6,653,124 “Array-based microenvironment for cell    culturing, cell monitoring and drug-target validation.”-   Cellomics, Inc. 6,548,263 “Miniaturized cell array methods and    apparatus for cell-based screening.”-   Fluidigm, Inc. Published Application 20040229349 (Nov. 18, 2004)    “Microfluidic particle-analysis systems.”

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Earlier work and patent applications as cited above involving at leastone of the present inventors discuss various configurations, methods,and systems related to microfluidic cell culture and that work isincorporated herein by reference.

SUMMARY

The present invention involves various components, systems, and methodsrelated to improved microfluidic cell culture systems. In one aspect,the invention involves novel microfluidic cell culture systems andmethods that have advantages over previously proposed microfluidicstructures. In another aspect, the invention involves novel structuresand methods for integrating multiple microfluidic cell culture systemsto a microtiter well plate structure, such as a standard culture-wellplate formats (e.g., a 96-well SBS culture plate). In a further aspect,the invention involves novel fabrication methods for creating an arrayof microfluidic cell culture areas suitable for integration with a wellplate. In another aspect, the invention involves novel systems, methods,and components for an improved automated high-throughput cell cultureand/or screening system using microfluidic cell cultures.

In particular embodiments, key design features include the eliminationof tubing and connectors to the plates themselves, the ability tomaintain long-term continuous perfusion cell culture using a passivegravity-driven flow, and direct analysis on the outlet wells and/orcellular observation wells of the microfluidic plate.

For purposes of clarity, this discussion refers to devices, methods, andconcepts in terms of specific examples. However, the invention andaspects thereof may have applications to a variety of types of devicesand systems. It is therefore intended that the invention not be limitedexcept as provided in the attached claims and equivalents.

Furthermore, it is well known in the art that systems and methods suchas described herein can include a variety of different components anddifferent functions in a modular fashion. Different embodiments of theinvention can include different mixtures of elements and functions andmay group various functions as parts of various elements. For purposesof clarity, the invention is described in terms of systems that includemany different innovative components and innovative combinations ofinnovative components and known components. No inference should be takento limit the invention to combinations containing all of the innovativecomponents listed in any illustrative embodiment in this specification.

In some of the drawings and detailed descriptions below, the presentinvention is described in terms of the important independent embodimentof a complete, fully automated, cellular culture system and componentsthereof. This should not be taken to limit various novel aspects of theinvention, which, using the teachings provided herein, can be applied toa number of other situations. In some of the drawings and descriptionsbelow, the present invention is described in terms of a number ofspecific example embodiments including specific parameters related todimensions of structures, pressures or volumes of liquids, temperatures,electrical values, and the like. Except where so provided in theattached claims, these parameters are provided as examples and do notlimit the invention to other devices or systems with differentdimensions. For purposes of providing an more illuminating description,particular known fabrication steps, cell handling steps, reagents,chemical or mechanical process, and other known components that may beincluded to make a system or manufacture a device according to specificembodiments of the invention are given as examples. It will beunderstood to those of skill in the art that except were specificallynoted herein otherwise, various known substitutions can be made in theprocesses described herein.

All references, publications, patents, and patent applications cited inthis submission are hereby incorporated by reference in their entiretyfor all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top view of an example array of cell culture units accordingto specific embodiments of the invention. In this example, 32 cultureunits are provided on a 96-well plate (such as the Society forBiomolecular Screening (SBS) standard microfluidic bioreactor arrayschematic), with wells arranged in 12 columns (shown vertically) by 8rows. In this example, each cell culture unit occupies three wells, onefor use as a medium inlet, one for use as a cell inlet/medium outlet,and one for use for cell imaging (which appears as a dark rectangle inthe wells in the figure) and/or for providing air passages to a cellculture area. In specific embodiments, each unit can be used as anindependent biomimetic cell.

FIG. 2 is an underside view showing one culture unit occupying threewells according to specific embodiments of the invention.

FIG. 3 is a close-up underside view illustrating details of themicrofluidic cell culture areas described above according to specificembodiments of the invention. In this figure, the cell inlet/mediaoutlet is to the left, and the media inlet is to the right.

FIG. 4 is a close up micrograph of a cell culture area illustrating twolarge air holes at the left of the figure each connected to an airpassage that is placed between the blocks, each block having four cellculture sinusoids according to specific embodiments of the invention. Inthis figure, the cell inlet/media outlet is to the right, and the mediainlet is to the left. Also visible in the photo, are a media multiplexorstructures to the left in each block, and an optional cell inletmultiplexor to the right in each block.

FIG. 5 is a close up micrograph of a cell culture area illustrating twolarge air holes at the left of the figure each connected to an airpassage that is placed between the blocks, each block having eight cellculture sinusoids according to specific embodiments of the invention.

FIG. 6 illustrates high aspect ratio channels surrounding cell cultureareas wherein channels between solid structures are approximately 4 μmwide and 40 μm tall to prevent cells from growing out. The channels inthis example are separated by approximately 40 μm.

FIG. 7A illustrates a cell inlet/media outlet of a modified cell culturearea with a large rectangular cell inlet to provide for easier cellloading and with a cell loading perfusion area and a solid wall cellculture area. The arrows from the right indicate cell-loading direction.

FIG. 7B illustrates the media inlet/cell culture area of a modifiedmicrofluidic cell culture system according to specific embodiments ofthe invention. In this example, cell loading is from the right and mediaflow, as indicated by the arrows, is from the left.

FIG. 8 is a schematic showing three blocks of four long cell culturesinusoids, where the long cell sinusoids extend across two wells, andfurther shows a rectangular cell inlet region/flow outlet region, andfour air holes connecting to four air channels.

FIG. 9A-B are simplified schematic diagrams illustrating in threedimensions the components of a multi cell (e.g., 3) microfluidic systemincluding a representation of the well frame according to specificembodiments of the invention.

FIG. 10 is a simplified side view showing a structure according tospecific embodiments of the invention illustrating two wells that areused in cell flow and fluid flow.

FIG. 11 is a close-up micrograph showing cells loaded in five sinusoidcell culture regions with four sinusoid channels between according tospecific embodiments of the invention.

FIG. 12 shows four close-up micrographs showing cells loaded in fourdifferent sized sinusoid cell culture regions according to specificembodiments of the invention.

FIG. 13 is a schematic diagram showing steps from an empty cultureregion to performing a cell assay according to specific embodiments ofthe invention.

FIG. 14A-C shows a top view, side view, and plan view of a schematic ofan example manifold according to specific embodiments of the invention.In this example, the eight tubing lines to the right are for compressedair, and each is configured to provide pressure to a column of cellinlet wells in a microfluidic array. The left-most line in the figure isfor vacuum and connects to an outer vacuum ring around the manifold.Each column of wells is generally connected to a single pressure linewith wells above imaging regions skipped.

FIG. 15 is a graph illustrating an example of flow rate differencebetween a surface tension mechanism and a gravity driven mechanismaccording to specific embodiments of the invention.

FIG. 16 is a graph illustrating an example of the extent to whichgravity perfusion rate is responsive to the liquid level differencebetween the two upper reservoir wells according to specific embodimentsof the invention.

FIG. 17A-B illustrate a tilting platform that can be used to control theliquid height difference between the inlet/outlet wells in a device orsystem according to specific embodiments of the invention and an exampleof flow rate versus plate tilt angle.

FIG. 18 illustrates a top view schematic of an example cell cultureautomation system according to specific embodiments of the invention.

FIG. 19 is a photograph of an example automated microfluidic perfusionarray system according to specific embodiments of the invention.

FIG. 20 is a flow chart illustrating the process flow of typicaloperation steps.

FIG. 21 illustrates four microfluidic culture areas from an examplearray plate prepared in the example system described above using primaryrat hepatocytes.

FIG. 22 illustrates a portion of a microfluidic culture area from anexample array plate prepared in the example system described above usingprimary human hepatocytes.

FIG. 23 illustrates a layout of another type of cell culture arraydesigned for general cell culture automation according to specificembodiments of the invention.

FIG. 24 illustrates an operation schematic for performing automated cellculture and immunostaining on a microfluidic array with gravity cellloading as described above. Example applications include stem cellculture and primary cell culture with immunofluorescence staining andmicroscopy.

FIG. 25 illustrates an alternative example SBS (Society for BiomolecularScreening) standard microfluidic bioreactor array schematic.

FIG. 26A-B illustrate an alternative cellular culture system assemblyaccording to specific embodiments of the present invention showing (A)an example schematic microfluidics design for three cell units; (B) asoft lithography fabrication of this design with laser machining of fouropenings per culture unit.

FIG. 27 illustrates operation steps of a less automated or prototypesystem according to specific embodiments of the invention.

FIG. 28 illustrates a microfluidic mold fixed on glass plate withmagnets according to specific embodiments of the invention. In oneexample embodiments, each magnet used is: ¾″ Diameter× 1/16″ Thickfabricated, e.g., from sintered Neodymium-Iron-Boron (NdFeB) with aPlating/Coating of Ni—Cu—Ni (Nickel) and a Grade: N40 with a Pull Forceof: 4.0 lbs (1814 g).

FIG. 29 illustrates a stack of molds held together with magnetic clamps(e.g., Stack of self-aligned microfluidic molds) for forming polymermicro-molded structures according to specific embodiments of theinvention.

FIG. 30 is a block diagram showing an example direct soft moldingprocess according to specific embodiments of the invention.

FIG. 31A illustrates the two pieces in position before mounting to glassand coating. Because the top piece is injection molded, the bottom ofthe wells can be flat, rounded or tapered. One particular desiredfeature is that the bottom of the top piece, which covers themicrofluidic structures, should be flat to ensure uniform molding acrossthe array. This top piece can be either a proprietary top piece withwells as shown or, alternatively, can be a standard SBS multi-wellplate.

FIG. 31B illustrates an example wherein the bottom of the top piece ischemically modified by a reagent (Sylgard Primecoat) so the softpolymers adhere to the bottom of the top piece after the molding processand illustrates an appropriate amount of soft polymer poured onto thecenter of the mold (usually a few milliliters, depending on the area tobe covered as well as the thickness of the soft polymer after molding).

FIG. 31C illustrates an example wherein the top piece and the mold aresandwiched between two pieces of flat surfaces (usually glass plates)with clamping mechanisms (in this case, magnets) and the clampingmechanism holds the top piece and the mold together with alignment marksfitted to each other.

FIG. 31D illustrates an example wherein after detaching the moldedmicrofluidic cell culture array with the top piece, a laser cutter isused to create fluidic connections between the microfluidic structuresand the wells at specific locations (cell/reagent inlets/outlets).

FIG. 31E illustrates an example wherein the microfluidic cell culturearray is bonded to a piece of rectangular glass. The glass and/or arraymay be subjected to oxygen plasma treatment before the bonding.

FIG. 31F illustrates an example wherein using a liquid dispenser, themicrofluidic cell culture array is filled with priming solutions tomaintain its modified surface chemistry. If bubbles appear to be trappedinside the array, additional vacuum steps are used to eliminate thebubbles.

FIG. 31G illustrates an example wherein to prevent liquid evaporation,the array is sealed with a tape.

FIG. 31H illustrates an example wherein the array is optionally fit intoa frame so the finished array can be treated like a standard microtiterplate with the correct outside dimensions. In the case where the toppiece is a standard microtiter well plate, this step may be unnecessary.

FIG. 32A-D illustrate four components of a direct soft molding processaccording to specific embodiments of the invention.

FIG. 33A illustrates a step wherein an appropriate amount of softpolymer is poured onto the center of the mold (usually a fewmilliliters, depending on the area to be covered as well as the spacerthickness) For example, for a mold 6″ in diameter and a 150 micronspacer, the minimum amount required is π×7.62 cm×7.62 cm×0.015 cm˜2.75mL).

FIG. 33B illustrates a step wherein the acrylic sheet is sandwichedbetween two pieces of the glass plates so the magnets will press theacrylic sheet (with primer modified surface facing the mold) against themold until the acrylic sheet hits the spacer. The soft polymer will thenfill the space between the acrylic sheet and the mold to replicate themicrofluidic structures. In particular embodiments, the magnet-assistedclamping mechanism holds the pieces together while the soft polymer iscured at elevated temperature (60 degree C.) for at least 2 hours.

FIG. 33C illustrates that after cooling the compartments down toapproximately room temperature, the acrylic sheet with the soft polymeris detached from the mold. The microfluidic cell culture array istruthfully molded onto the soft polymer. To protect the soft polymersurface from contaminations from following processes, a surfaceprotection tape (Ultron Blue Adhesive Plastic Film 80 micron) may beapplied to the top of the surface of the elastomer by a roller.

FIG. 33D illustrates that after separation from the mold a CO2 lasercutter (VersaLaser, 25W model) is used to create fluidic connectionsbetween the microfluidic structures and the injection molded wells (cellinlet and medium inlet). Since the soft polymer used in the process isgas permeable, “air holes” may be cut near the cell culture areas topromote air diffusion for better cell culture. The circular top piece ismay be trimmed to rectangular shape at this stage.

FIG. 33E illustrates that after the surface protection tape is removedand the array is optionally ultrasonically cleaned (or water-jetcleaned) to shake off any dust created by the laser cutting step andoptionally a new surface protection tape is applied, the microfluidiccell culture array is glued to the injection molded plate with anultra-violet (UV) curable glue which is also bio-compatible (Loctite3301). The plate with the microfluidic cell culture array is cured in aUV chamber for 30 minutes. After removal of the surface protection tape,both a glass substrate (e.g., White Float Glass) and the microfluidiccell culture array undergo oxygen plasma treatment to activate thesurface and the glass substrate encloses the microfluidic cell culturearray through covalent bonding, as shown in FIG. 33F.

FIG. 33G illustrates that using a liquid dispenser, the microfluidiccell culture array is filled with priming solutions, as bubbles may beinside the array; and the array may be placed inside a vacuum chamberfor bubbles removal and may also be placed inside a UV/Ozone chamber(Novascan) for sterilization.

FIG. 33H illustrates that to prevent liquid evaporation, the array issealed with a tape (Excel Scientific, AlumaSeal).

FIG. 34 is a block diagram showing a representative example logic devicein which various aspects of the present invention may be embodied.

FIG. 35 (Table 1) illustrates an example of diseases, conditions, orstates that can evaluated or for which drugs or other therapies can betested according to specific embodiments of the present invention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS 1. Overview DEFINITIONS

A “particle” refers to biological cells, such as mammalian or bacterialcells, viral particles, or liposomal or other particles that may besubject to assay in accordance with the invention. Such particles haveminimum dimensions between about 50-100 nm, and may be as large as 20microns or more. When used to describe a cell assay in accordance withthe invention, the terms “particles” and “cells” may be usedinterchangeably.

A “microwell” refers to a micro-scale chamber able to accommodate aplurality of particles. A microwell is typically cylindrical in shapeand has diameter and depth dimensions in a preferred embodiment ofbetween 100 and 1500 microns, and 10 and 500 microns, respectively. Whenused to refer to a microwell within the microwell array device of theinvention, the term “well” and “microwell” are used interchangeably.

A “microchannel” refers to a micron-scale channel used for connecting astation in the device of the invention with a microwell, or a stationand a valve associated with the microwell. A microchannel typically hasa rectangular, e.g., square cross-section, with side and depthdimensions in a preferred embodiment of between 10 and 500 microns, and10 and 500 microns, respectively. Fluids flowing in the microchannelsmay exhibit microfluidic behavior. When used to refer to a microchannelwithin the microwell array device of the invention, the term“microchannel” and “channel” are used interchangeably.

A “microfluidics device” refers to a device having various station orwells connected by micron-scale microchannels in which fluids willexhibit microfluidic behavior in their flow through the channels.

A “microwell array” refers to an array of two or more microwells formedon a substrate.

A “device” is a term widely used in the art and encompasses a broadrange of meaning. For example, at its most basic and least elaboratedlevel, “device” may signify simply a substrate with features such aschannels, chambers and ports. At increasing levels of elaboration, the“device” may further comprise a substrate enclosing said features, orother layers having microfluidic features that operate in concert orindependently. At its most elaborated level, the “device” may comprise afully functional substrate mated with an object that facilitatesinteraction between the external world and the microfluidic features ofthe substrate. Such an object may variously be termed a holder,enclosure, housing, or similar term, as discussed below. As used herein,the term “device” refers to any of these embodiments or levels ofelaboration that the context may indicate.

Microfluidic systems provide a powerful tool to conduct biologicalexperiments. Recently, elastomer-based microfluidics has especiallygained popularity because of its optical transparency, gas permeabilityand simple fabrication methods. However, the interface with theend-users requires labor-intensive hole punching through the elastomer,and additional steps of tubing and syringe pump connection.

The present invention involves integrated elastomer-based microfluidicson standard well plates, with special focus on hepatocyte cultureapplications. The invention further involves methods of manufacture ofsuch plates and components and a system for automating cell cultureusing such plates. Advantages of specific embodiments include use of astandard microtiter plate format, tubing free cell culture, and abiomimetic liver microenvironment.

A system according to specific embodiments of the invention (forexample, using 96-well standard plates) can be operated using standardtechniques and equipment for handling standard microtiter plates, as arewell known in the art. For example, liquid dispensing is achieved withstandard pipette mechanics, and cell culture and analysis can be madecompatible with existing incubators and plate readers.

According to further embodiments of the invention, a novel cell loadingsystem uses a pneumatic manifold and pneumatic pressure to place cellsin the micro culture area. With the addition of this cell loadingsystem, microfluidic cell culture and analysis can be fully automatedusing other automated equipment that exists for handling standard titerplates.

In further embodiments, the gravity driven flow culture configurationutilizes the medium level difference between the inlet and outlet wellas well as engineering the fluidic resistances to achieve the desirableflow rate in nL/min regime. This provides the significant advantage ofbeing able to “passively” flow culture medium for long periods of time(up to 4 days) without the use of bulky external pumps or tubes.

In further embodiments, the invention involves a microfluidic system toallow control of the cell culture environment for long-term time-lapsemicroscopy of adherent cells. As the trend towards “systems biology”continues, it will become increasingly important to study dynamicbehavior in individual live cells as well as to improve thefunctionality and economics of high throughput live cell screening.According to specific embodiments of the invention, the inventionprovides a multiplexed microfluidic flow chamber allowing for time-lapsemicroscopy experimentation among other assays. The microfluidic chamberuses an artificial endothelial barrier to separate cells from flowchannels. The device is formatted to a standard well plate, allowingliquid and cell samples to be directly pipetted into the appropriateinlet reservoirs using standard equipment. A custom pneumatic flowcontroller is then used to load the cells into the culture regions aswell as to switch between different exposure solutions. A digitalsoftware interface can be used to allow a user to program specificinputs (pulses, ramps, etc.) over time to expose the cells to complexfunctions during time-lapse imaging.

Dynamic responses in living cells are the foundation for phenomena suchas biological signal processing, gene expression regulation,differentiation, and cell division. In specific embodiments, theinvention involves a system capable of controlling the cellularmicro-environment in a multiplexed format compatible with current cellculture methods. Cell response can be quantified using highmagnification fluorescence microscopy to derive kinetic information withsub-cellular resolution. This capability has broad applications incellular systems biology where dynamic single cell response experimentsare not currently practical.

2. Microfluidic Culture System and Array

The application referenced above (U.S. Ser. No. 11/994,997) discussed avariety of different cell culture configurations and fabricationtechniques. Portions of the operation of the cell culture areas andmaterials are useful as background to the present discussion. In someexamples therein, one or more micro culture areas are connected to amedium or reagent channel via a grid of fluidic passages (or diffusioninlets or conduits), wherein the grid comprises a plurality ofintersection micro high fluidic resistance passages. In one discussedexample, passages in the grid are about 1 to 4 μm in height, 25 to 50 μmin length and 5 to 10 μm in width, the grid allowing for more evendiffusion between medium or reagent channels and the culture area andallowing for easier manufacturing and more even diffusion. The earlierapplication further discussed that the high fluidic resistance ratiobetween the microchamber and the perfusion/diffusion passages or grid(e.g., ratios in the range of about 10:1, 20:1 to 30:1) offers manyadvantages for cell culture such as: (1) size exclusion of cells; (2)localization of cells inside a microchamber; (3) promoting a uniformfluidic environment for cell growth; (4) ability to configure arrays ofmicrochambers or culture areas; (4) ease of fabrication, and (5)manipulation of reagents without an extensive valve network. Exampleswere illustrated wherein a grid-like perfusion barrier can be muchshorter than the culture area or can be near to or at the same height,according to specific embodiments of the invention and further whereinvarious configurations for culture devices were illustrated. Theapplication also discussed a CAD drawing of a proposed 96-unitmicrofluidic bioreactor wherein each well was an SBS standard size (3.5mm in diameter) in order to be compatible with existing robotic liquidhandling systems and plate readers. The application also discussedseveral different configurations for an artificial sinusoid using bothcut passages and grids and with a flow-around perfusion design.

FIG. 1 is a top view of an example array of cell culture units accordingto specific embodiments of the invention. In this example, 32 cultureunits are provided on a 96-well plate (such as the Society forBiomolecular Screening (SBS) standard microfluidic bioreactor arrayschematic), with wells arranged in 12 columns (shown vertically) by 8rows. In this example, each cell culture unit occupies three wells, onefor use as a medium inlet, one for use as a cell inlet/medium outlet,and one for use for cell imaging (which appears as a dark rectangle inthe wells in the figure) and/or for providing air passages to a cellculture area. In specific embodiments, each unit can be used as anindependent biomimetic cell.

FIG. 2 is an underside view showing one culture unit occupying threewells according to specific embodiments of the invention. In thisexample, the cell culture portion visible in the middle well is dividedinto four blocks, with each block having four separated cell culturechannels surrounded by medium channels used for medium fluidic passage.In particular embodiments, these four separated cell culture channelsmay be referred to as sinusoids or artificial sinusoids, regardless ofwhether the far end of the areas has a rounded shape. Separation intofour blocks facilitates air diffusion through the material that definesthe microfluidic channels (such as silicone elastomerpolydime-thylsiloxane (PDMS)) structure into the culture areas. Six airholes to facilitate air passage are shown.

FIG. 3 is a close-up underside view illustrating details of themicrofluidic cell culture areas described above according to specificembodiments of the invention.

FIG. 4 is a close up micrograph of a cell culture area illustrating twolarge air holes at the left of the figure each connected to an airpassage that is placed between the blocks, each block having four cellculture sinusoids according to specific embodiments of the invention.

FIG. 5 is a close up micrograph of a cell culture area illustrating twolarge air holes at the left of the figure each connected to an airpassage that is placed between the blocks, each block having eight cellculture sinusoids according to specific embodiments of the invention.

FIG. 6 illustrates high aspect ratio channels surrounding cell cultureareas wherein channels between solid structures are approximately 4 μmwide and 40 μm tall to prevent cells from growing out. The channels inthis example are separated by approximately 40 μm.

FIG. 7A illustrates a cell inlet/media outlet of a modified cell culturearea with a large rectangular cell inlet to provide for easier cellloading and with a cell loading perfusion area and a solid wall cellculture area.

FIG. 7B illustrates the media inlet/cell culture area of a modifiedmicrofluidic cell culture system according to specific embodiments ofthe invention. In this example, cell loading is from the right and mediaflow, as indicated by the arrows, is from the left. A further differencein the modified design is that perfusion passages are absent in aportion of the cell culture channel (or artificial sinusoid). This hasbeen found to more easily locate cells at the end of the cell culturechannel in the cell culture area. Optionally, a portion of the cellculture channel near the fluid outlet has perfusion passages to ensurefluid flow after cells have aggregated at the culture end. The improveddesign provides for easier cell loading and a longer cell culture areasand cell culture channels to culture more cells and more uniform flow ofnutrients. It has been found that in operation cells localize/stick tothe areas of the culture channel that are immediately next to theperfusion passages. The segment of the cell culture channel between themain culture area region and the other set of perfusion passages nearthe cell inlet is devoid of cells, because the flow profile carries themout, particularly during cell loading. Thus, this modified designprevents the cells from spreading into the “flow” channels after a fewdays and stop the flow. In the modified design, the flow remainsunhindered since the cells cannot spread past the long cell culturechannel segment (where there are no perfusion passages). In an examplesystem, up to about 2,500 liver cells may be cultured in each area asshown in FIG. 7 and FIG. 8.

FIG. 8 is a schematic showing three blocks of four long cell culturesinusoids, where the long cell sinusoids extend across two wells, andfurther shows a rectangular cell inlet region/flow outlet region, andfour air holes connecting to four air channels.

FIG. 9A-B are simplified schematic diagrams illustrating in threedimensions the components of a multi cell (e.g., 3) microfluidic systemincluding a representation of the well frame according to specificembodiments of the invention.

FIG. 10 is a simplified side view showing a structure according tospecific embodiments of the invention illustrating two wells that areused in cell flow and fluid flow.

FIG. 11 is a close-up micrograph showing cells loaded in five sinusoidcell culture regions with four sinusoid channels between according tospecific embodiments of the invention.

FIG. 12 shows four close-up micrographs showing cells loaded in fourdifferent sized sinusoid cell culture regions according to specificembodiments of the invention.

Thus, the present invention according to specific embodiments of theinvention provides a number of novel improved microfluidicconfigurations. In a first aspect, three wells are used for eachotherwise independent cell culture system. In a second aspect,artificial sinusoids with artificial epithelial barriers are providedwith just one (optionally shared or multiplexed) fluidic inlet and one(optionally shared or multiplexed) fluidic output, where the mediumoutput also functions as a cellular input. In a third aspect, artificialsinusoids with artificial epithelial barriers with just one fluidicinlet and one fluidic output are divided into blocks with air channelsprovided between blocks. In a fourth aspect, air holes are provided inthe well chamber above the cell culture area of a microfluidic cellularculture array, where the medium output also functions as a cellularinput. In a fifth aspect, a multiplexed medium inlet structure andmultiplexed cellular input structure are provided to connect inputs andoutputs to blocks of artificial sinusoids. In a sixth aspect, amultiplexed medium inlet structure and larger shared cellular inputstructure are provided to connect inputs and outputs to blocks ofartificial sinusoids. In a seventh aspect, artificial sinusoids areconfigured with non-open portions of an epithelial barrier to betterlocalize cells, and with perfusions inlets surrounding a cell culturearea and optionally also present near a cell inlet area of the sinusoid.In an eighth aspect, longer artificial sinusoid chambers are provided.

As discussed elsewhere, various modifications may be made to thecell-culture area as described above. Various configurations arepossible for the epithelial barrier, such as a grid-like passagestructure. Other variations will be suggested to those of skill in theart having the teachings provided herein.

The structures disclosed above can also be adapted to systems using moreor fewer wells on a standard microtiter well plate, such as thosedescribed in referenced documents and in other examples herein.

3. Example Device Operation

FIG. 13 is a schematic diagram showing steps from an empty cultureregion to performing a cell assay according to specific embodiments ofthe invention. Various novel aspects according to specific embodimentsof the invention simplify these steps and allow them to be automated.

Cell Loading

Cell loading in specific embodiments of the invention can utilize therapid surface tension flow between the cell inlet and the flow inlet. Inthis method, the cell inlet reservoir (upper and lower) is aspirated ofits priming solution. Then, the flow inlet upper reservoir is aspirated.An amount (e.g., Five microliters) of cell suspension (e.g., trypsinizedHeLa human cancer cell line, 5×10̂5 cells/ml) is dispensed into the cellinlet lower reservoir. The flow inlet lower reservoir is aspirated,causing liquid to flow from cell inlet to flow inlet via surfacetension/capillary force. Cell loading in various configurations can becompleted in approximately 2-5 minutes. The cell loading reservoir isthen washed with medium (e.g., Dulbecco's Modified Eagle's Medium, DMEM)and filled with e.g., 50-100 microliters of clean medium. At this state,the plate is was placed in a controlled culture environment for a period(e.g., 37° C., 5% CO₂ incubator for 2-4 hours) to allow for cellattachment.

While such loading is effective for some microfluidic cell culturedevices, in a presently preferred embodiment, a proprietary pneumaticmanifold, as described elsewhere herein, is mated to the plate andpneumatic pressure is applied to the cell inlet area for more effectivecell loading. For particular cell systems, it has been found thatoverall cell culture area design can be made more effective when it isnot necessary to allow for passive cell loading.

FIG. 14A-C shows a top view, side view, and plan view of a schematic ofan example manifold according to specific embodiments of the invention.In this example, the eight tubing lines to the right are for compressedair, and each is configured to provide pressure to a column of cellinlet wells in a microfluidic array. The left-most line in the figure isfor vacuum and connects to an outer vacuum ring around the manifold.Each column of wells is generally connected to a single pressure linewith wells above imaging regions skipped. The manifold is placed on topof a standard well plate. A rubber gasket lies between the plate andmanifold, with holes matching the manifold (not shown). The vacuum linecreates a vacuum in the cavities between the wells, holding the plateand manifold together. Pressure is applied to the wells to drive liquidinto the microfluidic channels (not shown). A typical pressure of 1 psiis used, therefore the vacuum strength is sufficient to maintain anair-tight seal. In one example there are 9 tubing lines to the pressurecontroller: 8 lines are for compressed air and 1 line is for vacuum(leftmost). In specific example embodiments, each column is connected toa single pressure line. Columns above the cell imaging regions areskipped.

Pressurized cell loading in a system according to specific embodimentsof the invention has been found to be particularly effective inpreparing cultures of aggregating cells (e.g., solid tumor, liver,muscle, etc.). Pressurized cell loading also allows structures withelongated culture regions, e.g., as shown in FIG. 7 and FIG. 8, to beeffectively loaded. Use of a pressurized manifold for cell loading andpassive flow for perfusion operations allows the invention to utilize afairly simple two inlet design, without the need for additional inletwells and/or valves as used in other designs.

Fluid Flow and Operation: Gravity and Surface Tension Flow

The format of the microfluidic plate design allows twoautomation-friendly flow modalities dependent on the extent ofdispensing/aspiration. The first is surface tension mediated flow. Inthis case, when the lower reservoir is aspirated in either one of thewells, the capillary force of the fluid/air interface along with thewetted surfaces (glass, silicone, acrylic) will rapidly draw liquid infrom the opposing well until the lower reservoir is filled (or inequilibrium with the opposing lower reservoir). This effect is usefulfor microfluidic flows as it is only evident when the reservoir diameteris small and the flow volumes are small. In an example array design, thelower reservoir wells are 1-2 mm in diameter, and with a total flowvolume of approximately 3-5 microliters. Since the microfluidic channelvolume is only 0.2 microliters, this mechanism is well suited for cellloading and cell exposures.

The second mechanism is gravity driven perfusion, which is well suitedfor longer term flows, as this is dependent on the liquid leveldifference and not the reservoir dimensions. According to specificembodiments of the invention, this may be accomplished by adding moreliquid into one reservoir (typically filling near the top of the upperreservoir). The fluidic resistance through the microfluidic channelswill determine how long (e.g., 24 hours) to reach equilibrium betweenthe wells and thus determine how often wells should be refilled.

FIG. 15 shows the flow rate difference between the surface tensionmechanism and the gravity driven mechanism. For the surface tensionflow, in an example, 5 microliters was dispensed into the lowerreservoir followed by aspiration of the opposing lower reservoir. Forthe gravity flow, a liquid level difference of 2.5 mm was used, withboth wells filled into the upper reservoir portion.

Changing Gravity Flow Rate via Liquid Level

The gravity perfusion rate is also responsive to the liquid leveldifference between the two upper reservoir wells as illustrated in FIG.16. This fact allows an automated dispenser/aspirator to control andmaintain a given perfusion flow rate over a 10-fold range duringculture. Here, different liquid level differences were produced viadispensing volumes and measured for volumetric flow rate.

Controlling Gravity Perfusion Rate via Plate Tilt Angle

According to specific embodiments of the invention, the liquid heightdifference between the inlet/outlet wells across the plate can also beprecisely controlled using a mechanical tilting platform. In thisimplementation, it is possible to maintain a constant flow rate overtime, as well as back-and-forth flow with different forward and reversetimes (i.e. blood flow). In the example illustrated in FIG. 17, bothinlet and outlet reservoirs were filled with 50 microliters of solution.On a flat surface, there is no flow through the channels, and as theangle is increased, so is the flow rate. The photo shows a prototypecontrolled tilting platform, consisting of a mechanical platform, and anelectronic switch.

In an example system, perfusion cell culture can be initiated by fillingthe flow inlet reservoir with 200-300 microliters of fresh medium (e.g.,DMEM supplemented with 10% fetal bovine serum) and aspirating the cellinlet upper reservoir. The liquid level difference between the flowinlet and cell inlet wells will then cause a continuous gravity drivenflow through the attached cells. For sustained culture, the flow inletwell is refilled and the cell inlet well aspirated during a perioddepending on fluidic resistance and reservoir volumes (e.g., every 24hours).

Cell Assay and/or Observation

Cell assay can be performed directly on the microfluidic cell cultureusing standard optically based reagent kits (e.g. fluorescence,absorbance, luminescence, etc.). For example a cell viability assayutilizing conversion of a substrate to a fluorescent molecule by livecells has been demonstrated (CellTiter Blue reagent by PromegaCorporation). The reagent is dispensed into the flow inlet reservoir andexposed to the cells via gravity perfusion over a period of time (e.g.,21 hours). For faster introduction of a reagent or other fluid, the newfluid can be added to the flow inlet reservoir followed by aspiration ofthe cell inlet reservoir.

Data can be collected directly on the cells/liquid in the microfluidicplate, such as placing the plate into a standard fluorescence platereader (e.g., Biotek Instruments Synergy 2 model). In some reactions,the substrate may diffuse into the outlet medium, and therefore beeasily detected in the cell inlet reservoir. For cell imaging assays,the plate can be placed on a scanning microscope or high content system.For example, an automated Olympus IX71 inverted microscope station canbe used to capture viability of cultured liver cells with a 20×objective lens.

By repeatedly filling/aspirating the wells, cells can be maintained forlong periods of time with minimal effort (e.g. compared to standard“bioreactors” which require extensive sterile preparation of large fluidreservoirs that cannot be easily swapped out during operation).

4. Automated Systems

FIG. 18 illustrates a top view schematic of an example cell cultureautomation system according to specific embodiments of the invention.Because the plates are designed to be handled using SBS compliantinstruments, various “off-the-shelf” machines can be used to create anautomated system. This schematic shows an example of how this isaccomplished. A robotic arm (plate handler) moves the microfluidicplates from station to station. An automated incubator stores the platesat the proper temperature and gas environment for long term perfusionvia gravity flow. The pipettor dispenses liquids (media, drugs, assayreagents, etc.) to the inlet wells and removes liquid from the outletwells. A plate reader is used for assay. The cell loader is optionallyused to introduce the cells to the microfluidic arrays at the beginningof the experiment. The cell loader in particular is generally not“off-the-shelf” and operates by applying pneumatic pressure to specifiedwells of the array plate to induce flow. Standard or custom computersoftware is available to integrate operations.

FIG. 19 is a photograph of an example automated microfluidic perfusionarray system according to specific embodiments of the invention. Thebasic process includes: 1) removing the plate from the incubator, 2)removing liquid from the outlet wells via the pipettor, 3) moving amedia/drug storage plate from the “plate stacks,” 4) transferring liquidfrom the media/drug plate to the microfluidic plate via the pipettor, 5)placing the microfluidic plate into the incubator, 6) repeat for eachplate, 7) repeat after specified time interval (e.g. 24 hours).

FIG. 20 is a flow chart illustrating the process flow of typicaloperation steps. This figure illustrates, as an example, automatedprocess steps an indicates an automated device that is used to performsuch a step. A standard automated pipettor is used for an optionalsurface coating, to add cells in suspension, to add media or drugs orreagents, and to change media. Known automated pipettors canindividually deliver or withdraw fluids from specified wells. In aspecific embodiment, a proprietary cell loader is used to pressurize thecell inlet wells for cell loading. After a period in an incubatordesigned for optimal cell attachment, the cell loader can again be usedto wash fluid and unattached cells from the microfluidic culture areas.One or more reading or analysis devices is used to assay the cells.

FIG. 21 illustrates four microfluidic culture areas from an examplearray plate prepared in the example system described above using primaryrat hepatocytes. The cells were cultured for 1 week with medium changedat a rate of 150 ul per unit, twice a day. Cells were assayed at the endof 7 days for viability using the Cell Titer Blue Kit from Promega, andread on an automated fluorescence plate reader (Biotek Synergy). In thisfigure, an example microfluidic culture area uses a grid flow-throughepithelial walls.

FIG. 22 illustrates a portion of a microfluidic culture area from anexample array plate prepared in the example system described above usingprimary human hepatocytes. The cells were cultured in the microfluidicarray according to specific embodiments of the invention, showing (a)phase contrast of freshly isolated human hepatocytes cultured in themicrofluidic device for 13 days. (b) Viability of human hepatocytescultured in the microfluidic device and in a 96-well dish measured bythe CellTiter Blue assay (Promega, Inc.). (c) P450 CYP3A4 activity ofcultured hepatocytes in the microfluidic device and 96-well dishmeasured via the P450-Glow assay (Promega, Inc.).

FIG. 23 illustrates a layout of another type of cell culture arraydesigned for general cell culture automation according to specificembodiments of the invention. In this design, each culture unit consistsof 4 well positions. The first well is for perfusion medium, the secondwell is for cell inlet, the third well is for imaging the microfluidicchamber, and the fourth well is the outlet. A cell barrier/perfusionbarrier localizes cells to the cell area and improves nutrient transportduring continuous perfusion culture. The low fluidic resistance of thecell inlet to outlet path enables cells to be rapidly loaded via gravityor surface tension methods without an external cell loading mechanism.The high fluidic resistance of the perfusion inlet channels allows longterm continuous perfusion of medium via gravity flow without anyexternal pump mechanism.

FIG. 24 illustrates an operation schematic for performing automated cellculture and immunostaining on a microfluidic array with gravity cellloading as described above. Example applications include stem cellculture and primary cell culture with immunofluorescence staining andmicroscopy.

FIG. 25 illustrates an alternative example SBS (Society for BiomolecularScreening) standard microfluidic bioreactor array schematic. A 16-unitmicrofluidic cell culture array filled with colored dyes so thatmicrofluidic channels are visible. In this example, each unit occupiesfive wells, which from left to right are medium inlet, cell inlet, celloutlet, cell imaging, and medium outlet.

FIG. 26A-B illustrate an alternative cellular culture system assemblyaccording to specific embodiments of the present invention showing (A)an example schematic microfluidics design for three cell units; (B) asoft lithography fabrication of this design with laser machining of fouropenings per culture unit. This design is attached to a microplate withwells for receiving medium and cells as described herein.

FIG. 27 illustrates operation steps of a less automated or prototypesystem according to specific embodiments of the invention. The 96-wellplate standard allows the microfluidic system to be operated usingstandard techniques and equipment. For example, liquid dispensing isachieved with standard pipette mechanics, and cell culture and analysisis compatible with existing incubators and plate readers. A custom builtcell loading system can be used to load the cells using air pressure asdescribed above. The gravity driven flow culture configuration utilizesthe medium level difference between the inlet and outlet well as well asengineering the fluidic resistances to achieve the desirable flow ratein nL/min regime. This provides the significant advantage of being ableto “passively” flow culture medium for long periods of time (forexample, up to 4 days) without the use of bulky external pumps.

FABRICATION TECHNIQUES 5. Example 1

FIG. 30 is a block diagram illustrating two components of a direct softmolding process according to specific embodiments of the invention. Thetwo components illustrated are: (1) An injection molded top piece madeof acrylic containing at least alignment marks (to be assembled to themicrofluidic mold) and well structures generally complying with standardmicrotiter plate formats. (Alternatively, a standard well plate may beused.) (2) A microfluidic mold fabricated using semiconductortechnologies on a 6″ silicon wafer containing the microfluidic cellculture arrays made of epoxy or electroplated metals, as well as thealignment marks so the well structures aligned to the microfluidicstructures during the molding process. An injection molded top piece ismade of acrylic or any similar suitable material and contains wellstructures that preferably comply with standard microtiter plate formatsas will be discussed more herein. On the right is shown a microfluidicmold fabricated using any known semiconductor and/or microfabricationtechnologies on, for example, a 6″ silicon wafer. The mold contains animpression the microfluidic cell culture arrays and can includecomponents made of epoxy or electroplated metals, as well as thealignment marks so the well structures aligned to the microfluidicstructures during the molding process. Generally, before furtherprocessing, the mold is coated with fluoropolymer to reduce stiction ofthe soft polymer to the mold.

Because the top piece containing the well structures is injectionmolded, the bottom of the wells can be flat, rounded or tapered. Oneparticular desired feature is that the bottom of the top piece, whichcovers the microfluidic structures, is as flat as practically to assistuniform molding across the array. According to specific embodiments ofthe invention, the bottom of the top piece can be chemically ormechanically or otherwise modified or primed by a reagent (such asSylgard Primecoat) or an abrasive surface (sanding) or laser so the softpolymers adhere to the bottom of the top piece after the moldingprocess. This treatment of the surface is indicated by the heavy line.

FIG. 31B illustrates an example wherein an appropriate amount of softpolymer is poured onto the center of the mold (usually a fewmilliliters, depending on the area to be covered as well as thethickness of the soft polymer after molding). The top piece and the moldare sandwiched between two pieces of flat surfaces (usually glassplates) with clamping mechanisms (in this case, magnets).

FIG. 31C illustrates an example wherein the clamping mechanism holds thetop piece and the mold together with alignment marks fitted to eachother. The soft polymer is then cured, for example by temperature or UVlight of otherwise so the microfluidic cell culture array is truthfullymolded onto the soft polymer. As an example, at elevated temperature(usually 60° C.) for at least 2 hours.

FIG. 31D illustrates an example wherein after detaching the moldedmicrofluidic cell culture array with the top piece, a laser cutter isused to create fluidic connections between the microfluidic structuresand the wells at specific locations (cell/reagent inlets/outlets). Thecircular top piece is trimmed to rectangular shape at this stage. (Notethat in this image, the structure is inverted.) Before enclosing thebottom of the microfluidic cell culture array, the molded piece isultrasonically cleaned to shake off any dust created by the lasercutting step. A top piece may be trimmed to a rectangular shape at thisstage. The cross section shown in through each of the fluidicconnections for illustration purposes, though the laser only makes holesin the material and does not cut the wells apart. At this state, beforeenclosing the bottom of the microfluidic cell culture array, the moldedpiece is preferably ultrasonically or otherwise cleaned to shake off anydust created by the laser cutting step.

FIG. 31E illustrates an example wherein the microfluidic cell culturearray undergoes oxygen plasma treatment and is bonded to a piece ofrectangular glass.

FIG. 31F illustrates an example wherein using a liquid dispenser, themicrofluidic cell culture array is filled with priming solutions tomaintain its modified surface chemistry. If bubbles appear to be trappedinside the array, placement in a vacuum chamber may be used to eliminatethe bubbles.

FIG. 31G illustrates an example wherein to prevent liquid evaporation,the array is sealed with a tape. FIG. 31H illustrates an example whereinthe array is fit into a frame so the finished array can be treated likea standard microtiter plate with the correct outside dimensions.

6. Example 2

FIG. 32A-C illustrate three components of a direct soft molding processaccording to specific embodiments of the invention. In the figure, (A)shows an injection molded top piece that includes well structurescomplying with standard microtiter plate formats. As discussed above,rather than the injection molded top piece shown, a standard microtiterplate may be used as the top piece that includes well structurescomplying with standard microtiter plate formats, (B) Illustrates a 1.5mm thick acrylic circular sheet (6″ in diameter), and (C) illustrates amicrofluidic mold fabricated on a 6″ silicon wafer containingmicrofluidic cell culture units (in this example 8×4 units) in an arraysmade of epoxy, etched silicon, or electroplated metals, as well as aspacer to control the minimum thickness of the soft polymer aftermolding. The mold is coated with fluoropolymer to reduce stiction of thesoft polymer to the mold. As shown in the figure, the microfluidic moldis glued to a 1 mm thick soda lime glass (7″×7″) with four magnets(e.g., 15 mm in diameter and 1.5 mm in thickness) glued to the fourcorners of the glass. The other piece of the 1 mm thick soda lime glass(7″×7″) with complementary magnets is prepared in similar fashion (FIG.32D) with the magnets of opposite polarity glued on the four corners sothe magnets will self-align onto the magnets in FIG. 32C. One side ofthe acrylic sheet is chemically modified by a reagent (e.g., SylgardPrimecoat) to induce the strong adhesion between the acrylic and thesoft polymer (e.g., Sylgard 184) to be used during the molding process.

FIG. 33A illustrates a step wherein an appropriate amount of softpolymer is poured onto the center of the mold (usually a fewmilliliters, depending on the area to be covered as well as the spacerthickness) For example, for a mold 6″ in diameter and a 150 micronspacer, the minimum amount required is π×7.62 cm×7.62 cm×0.015 cm˜2.75mL).

FIG. 33B illustrates a step wherein the acrylic sheet is sandwichedbetween two pieces of the glass plates so the magnets will press theacrylic sheet (with primer modified surface facing the mold) against themold until the acrylic sheet hit the spacer. The soft polymer will thenfill the space between the acrylic sheet and the mold to replicate themicrofluidic structures. In particular embodiments, the magnet-assistedclamping mechanism holds the pieces together while the soft polymer iscured at elevated temperature (60 degree C.) for at least 2 hours.

After cooling the compartments down to approximately room temperature,the acrylic sheet with the soft polymer is detached from the mold and amicrofluidic cell culture array as described herein is truthfully moldedonto the soft polymer.

To protect the soft polymer surface from contaminations from followingprocesses, a surface protection tape (e.g., Ultron Blue Adhesive PlasticFilm 80 micron) is optionally applied to the top of the surface of theelastomer by a roller.

FIG. 33D illustrates a step wherein a CO2 laser cutter (VersaLaser, 25Wmodel) is used to create fluidic connections between the microfluidicstructures and the injection molded wells (cell inlet and medium inlet).Since the soft polymer used in the process is gas permeable, “air holes”are cut near the cell culture areas to promote air diffusion for bettercell culture. The circular top piece may be trimmed to rectangular shapeat this stage. The surface protection tape is removed and the array isultrasonically cleaned (or water-jet cleaned) to shake off any dustcreated by the laser cutting step and a new surface protection tape isapplied. In FIG. 33E, the microfluidic cell culture array is glued tothe injection molded plate or a standard-well plate with an ultra-violet(UV) curable glue which is also bio-compatible (Loctite 3301). The platewith the microfluidic cell culture array is cured in a UV chamber for 30minutes. After removal of the surface protection tape, both a glasssubstrate (White Float Glass) and the microfluidic cell culture arrayundergo oxygen plasma treatment to activate the surface. The glasssubstrate encloses the microfluidic cell culture array through covalentbonding, as shown in FIG. 33F. Using a liquid dispenser, themicrofluidic cell culture array is filled with priming solutions asshown in FIG. 33G. Because bubbles may be present inside the array; thearray is generally placed inside a vacuum chamber for bubbles removal.The plate may also be placed inside a UV/Ozone chamber (Novascan) forsterilization. To prevent liquid evaporation, the array is sealed with atape (e.g., Excel Scientific, AlumaSeal) as shown in FIG. 33H.

Integrated Systems

Integrated systems for the collection and analysis of cellular and otherdata as well as for the compilation, storage and access of the databasesof the invention, typically include a digital computer with softwareincluding an instruction set for sequence searching and/or analysis,and, optionally, one or more of high-throughput sample control software,image analysis software, collected data interpretation software, arobotic control armature for transferring solutions from a source to adestination (such as a detection device) operably linked to the digitalcomputer, an input device (e.g., a computer keyboard) for enteringsubject data to the digital computer, or to control analysis operationsor high throughput sample transfer by the robotic control armature.Optionally, the integrated system further comprises valves,concentration gradients, fluidic multiplexors and/or other microfluidicstructures for interfacing to a microchamber as described.

Readily available computational hardware resources using standardoperating systems can be employed and modified according to theteachings provided herein, e.g., a PC (Intel x86 or Pentiumchip-compatible DOS,™ OS2,™ WINDOWS,™ WINDOWS NT,™ WINDOWS95,™WINDOWS98,™ LINUX, or even Macintosh, Sun or PCs will suffice) for usein the integrated systems of the invention. Current art in softwaretechnology is adequate to allow implementation of the methods taughtherein on a computer system. Thus, in specific embodiments, the presentinvention can comprise a set of logic instructions (either software, orhardware encoded instructions) for performing one or more of the methodsas taught herein. For example, software for providing the data and/orstatistical analysis can be constructed by one of skill using a standardprogramming language such as Visual Basic, Fortran, Basic, Java, or thelike. Such software can also be constructed utilizing a variety ofstatistical programming languages, toolkits, or libraries.

FIG. 34 shows an information appliance (or digital device) 700 that maybe understood as a logical apparatus that can read instructions frommedia 717 and/or network port 719, which can optionally be connected toserver 720 having fixed media 722. Apparatus 700 can thereafter usethose instructions to direct server or client logic, as understood inthe art, to embody aspects of the invention. One type of logicalapparatus that may embody the invention is a computer system asillustrated in 700, containing CPU 707, optional input devices 709 and711, disk drives 715 and optional monitor 705. Fixed media 717, or fixedmedia 722 over port 719, may be used to program such a system and mayrepresent a disk-type optical or magnetic media, magnetic tape, solidstate dynamic or static memory, etc. In specific embodiments, theinvention may be embodied in whole or in part as software recorded onthis fixed media. Communication port 719 may also be used to initiallyreceive instructions that are used to program such a system and mayrepresent any type of communication connection.

Various programming methods and algorithms, including genetic algorithmsand neural networks, can be used to perform aspects of the datacollection, correlation, and storage functions, as well as otherdesirable functions, as described herein. In addition, digital or analogsystems such as digital or analog computer systems can control a varietyof other functions such as the display and/or control of input andoutput files. Software for performing the electrical analysis methods ofthe invention are also included in the computer systems of theinvention.

Other Embodiments

Although the present invention has been described in terms of variousspecific embodiments, it is not intended that the invention be limitedto these embodiments. Modification within the spirit of the inventionwill be apparent to those skilled in the art.

It is understood that the examples and embodiments described herein arefor illustrative purposes and that various modifications or changes inlight thereof will be suggested by the teachings herein to personsskilled in the art and are to be included within the spirit and purviewof this application and scope of the claims.

All publications, patents, and patent applications cited herein or filedwith this submission, including any references filed as part of anInformation Disclosure Statement, are incorporated by reference in theirentirety.

1. A microfluidic cell culture system comprising: a medium inletreservoir fluidically connected to a plurality of microfluidic mediumchannels; a plurality of cell culture areas each located at an end of acell culture channel; said cell culture channel having a cell inletopening fluidically connected to a cell inlet reservoir for introducingcells to said plurality of cell culture areas; a perfusion barrierbetween said cell culture areas and said plurality of microfluidicmedium channels, said perfusion barrier containing a plurality ofperfusion passages; a media outlet reservoir fluidically connected tosaid cell culture channels; such that fluid introduced into said mediuminlet reservoir must pass through a part of said cell culture channelsbefore reaching said media outlet reservoir.
 2. (canceled)
 3. The systemof claim 1 further wherein said system is integrated into a standardwell plate and further wherein each independent device occupies threewell positions, a first well acting as a medium inlet, a second wellacting as a medium outlet/cell inlet, and a middle well acting as aviewing/cell culture area.
 4. The system of claim 1 further wherein saidcell culture channels include a first portion near said cell inlet thatcontains solid barriers walls with no perfusion passages to prevent celladhesion.
 5. The system of claim 1 further wherein said cell culturechannels include a second portion nearer to said cell inlet than saidfirst portion, said second portion having a plurality of perfusionpassages to facilitate media flow out of said cell culture region. 6.The system of claim 1 further wherein said standard well plate ishandled by robotic equipment allowing for automated cell culture andanalysis, said robotic equipment being in part equipment designed foruse with standard well plates.
 7. The system of claim 1 further whereinsaid standard well plate is configured to pneumatically couple with apneumatic manifold, said manifold providing pneumatic pressure to drivecells into said cell culture areas.
 8. The system of claim 1 furtherwherein: said cell culture areas are separated into at least threeblocks, each block containing at least two cell culture areas; eachblock having an air channel adjacent thereto.
 9. The system of claim 1further wherein: cells are introduced into said cell culture areas usingpneumatic pressure, and thereafter, cells are maintained by perfusion ofmedia using a passive gravity driven fluid flow.
 10. The system of claim1 further wherein: a plurality of said perfusion passages aresubstantially narrower than a diameter of cells to be cultured.
 11. Thesystem of claim 1 further wherein: a plurality of said perfusionpassages are substantially narrower than about 6 microns.
 12. The systemof claim 1 further comprising: a large fluidic opening directlyconnected to said cell culture channels to allow for easier cell flow.13. The system of claim 1 further comprising: a fluidic multiplexorconnecting said media inlet reservoir to said microfluidic mediumchannels.
 14. (canceled)
 15. The system of claim 1 further wherein: aplurality of said cell culture channels comprise a cell culture areawith perfusion passages to said media channels and a non perfusion cellchannel portion for better localizing cells introduced under pneumaticpressure and an outlet portion with perfusion passages near to an inletof said cell culture channel.
 16. The system of claim 1 further wherein:said cell culture areas are elongated areas mimetic of a liver sinusoid;said cell culture areas are loaded with cells from said cell inletreservoir; after cell loading and settling, media is loaded from saidmedia inlet and removed from said cell inlet/medium outlet reservoir.17. A microfluidic cell culture device comprising: a medium inletreservoir and a channel for introducing liquid media to a plurality ofcell culture areas; a cell inlet/medium outlet reservoir for introducingcells to said plurality of cell culture areas; wherein said cell cultureareas are elongated areas mimetic of a liver sinusoid; wherein said cellculture areas are loaded with cells from said cell inlet/medium outletreservoir; wherein after cell loading and settling, media is loaded fromsaid media inlet and received and/or removed from said cell inlet/mediumoutlet reservoir; such that a portion of said media passes through cellsin said culture area.
 18. (canceled)
 19. The device of claim 17 furtherwherein said device is integrated into a standard well plate and furtherwherein each independent device utilizes at least three well positions,a first well acting as a medium inlet, a second well acting as a mediumoutlet/cell inlet, and a middle well acting as a viewing/cell culturearea. 20-23. (canceled)
 24. The device of claim 17 further wherein: saidcell culture areas are separated into at least three blocks, each blockcontaining at least two cell culture areas; each block having an airchannel adjacent thereto.
 25. A method of culturing cells comprising:placing cells into a well of a standard well plate, said wellfluidically connected to a plurality of microfluidic channels, saidmicrofluidic channels connected to a plurality of cell culture areas;placing media into a different well of said standard well plate, saidwell fluidically connected to a plurality of microfluidic perfusionpassages, said microfluidic perfusion passages providing perfusionpassages through said micro cell culture areas; allowing said cells toculture for an appropriate time; observing and/or assaying cells and ormedia in said standard well plate.
 26. The method of claim 25 furthercomprising: interfacing said standard well plate with a pneumaticmanifold for providing air pressure to drive said cells into said cellculture areas, said pneumatic manifold providing sufficient pressure toform cell aggregates in said cell culture areas, wherein said pneumaticmanifold interfaces with said well plate using a vacuum seal. 27.(canceled)
 28. The method of claim 26 further comprising: using apipette to introduce and/or remove fluids from said reservoirs, saidfluids passively perfusing through said cell culture areas as a resultof gravity and/or differential fluid levels; and or surface tensions.29. (canceled)
 30. The method of claim 25 further wherein said steps areperformed by fully automated robotic equipment and further wherein saidcombination of pneumatic cell loading and passive perfusion allowssimultaneous automatic culture of a large number of plates using onepneumatic manifold and one automated pipettor because plates do not needto be attached to any equipment during perfusion fluid flow. 31-87.(canceled)